The biogenesis of organelles ensures propagation through successive generations but our knowledge of the underlying mechanisms is patchy, especially for membrane-bound organelles such as the Golgi apparatus. There are two popular models. The first considers the Golgi to be an autonomous organelle, responsible for its own duplication and partitioning during mitosis. This is the model we favor. The second model considers the Golgi to be a dependent organelle, in dynamic steady state with the ER, and reliant on it for all aspects of biogenesis. In the last granting period we studied those proteins that tether vesicles to Golgi membranes and cisternal membranes to each other. Collectively, we termed these matrix proteins and were able to provide evidence that they constituted an underlying scaffold that is normally populated by enzyme containing membranes. In this renewal we aim to confirm and extend these observations, focusing on the GRASP family of stacking proteins. Our aim is to determine precisely how these proteins carry out their stacking function. We also want to understand the process of Golgi assembly, both at the end of mitosis when the partitioned Golgi is reassembled, and during subsequent duplication, when a new copy of the Golgi is made. In particular we want to know whether the process is "seeded" by matrix proteins and whether there is a precise order of cisternal assembly. Lastly, we have begun to study the process of Golgi duplication in two protozoan parasites, Toxoplasma gondii, and Trypanosoma brucei. Both have a single Golgi apparatus, the duplication of which can be followed in real time using video fluorescence microscopy. These simple systems are also medically important. [unreadable] [unreadable]